The present invention relates to a method for implanting producer cells into the mammalian brain. The producer cells are engineered with a retroviral based recombinant vector encoding a tumorcidal factor or susceptibility factor that confers on tumor cells sensitivity to chemotherapeutic or radiotherapeutic agents. Prior to transplantation into the mammalian brain, the producer cells are first cultured in vitro on a support matrix to increase the long-term viability of the transplanted cells and to provide long-term functional benefit.
Brain tumors are the leading cause of cancer deaths in persons younger than 35 years. The incidence of central nervous system tumors is more than twice that of Hodgkin""s disease, more than half that of melanoma and, in women, the frequency of mortality caused by tumors of the central nervous system is almost equivalent to that caused by ovarian cancer. In children, brain tumors are the most common solid tumor and are second only to leukemia as an overall cause of childhood cancer. (Dale, D. C. and Federman, D. D., 1995, Scientific American Medicine, Scientific American, Inc., New York, Chapter 7.) Most brain tumors are inoperable; and even for those brain tumors that are operable, the surgery is extremely difficult and frequently leads to neurological disorders.
The in vivo application of retroviral vector-mediated gene therapy has been applied to the treatment of brain tumors (Oldfield et al., 1993, Hum. Gene Ther.; 4:39-69; Culver et al., 1992 Science 256:1550-2). Perhaps, the most widely studied application of gene therapy utilizes retroviruses genetically engineered to express proteins that activate a relatively nontoxic pro drug to form a highly toxic agent. For example, retroviral producer cells expressing susceptibility factors have been transplanted into the brain tissue of patients in order to kill the tumor cells (Barba, D. et al., WO 93/04167). One particular application of the system utilizes the thymidine kinase gene of the Herpes simplex virus which confers sensitivity to anti-viral drugs such as ganciclovir and acyclovir (Barba et al., WO 93/04167; Moolten, F. L. et al., 1986, Cancer Research 46:5276-5281). The HSV-TK gene product catalyzes the phosphorylation of a number of nucleoside analogues which are poor substrates for the TK of mammalian cells. For example, the antiherpes drug acyclovir exhibits minimal toxicity to cells lacking HSV-TK activity, but is activated in cells expressing HSV-TK to a toxic form capable of inhibiting DNA synthesis and which has been shown to exhibit selective cytoxicity to cells expressing the HSV-TK gene.
One concern associated with the use of retroviral vector-mediated gene therapy is that the implanted producer cells might not continue to survive and/or express the therapeutic genes for the time periods required to achieve the maximum therapeutic benefit. It is generally known that cells directly implanted into the brain die within about a two to four week period (see, for example, Itukura, T. et al., 1988, J. Neurosurg. 68:955-959). In some instances, the adherence of cells to microcarriers, prior to implantation in vivo, has been shown to enhance the long-term viability of transplanted cells (Cherskey et al.; WO 9206702) but to date this method has not been successfully applied to retroviral producer cell lines.
The present invention relates to a method for transferring genes encoding a tumoridal factor or susceptibility factor to brain tumor cells. The method comprises the implantation of producer cells engineered with a retroviral based recombinant vector encoding a tumorcidal factor or susceptibility factor into the mammalian brain. The engineered producer cells produce infectious retroviral particles which are capable of infecting the neighboring brain tumor cells thereby rendering the tumor cells sensitive to chemotherapeutic or radiotherapeutic agents. Since the retroviral vector gene transfer system requires a proliferating target cell for integration and gene expression in the brain, the application of this system to brain tumors has the advantage that the retroviruses are targeted to the proliferating cells of the brain tumor, while the normal non-proliferating brain cells remain uninfected.
A number of genes encoding tumorcidal or susceptibility factors may be used in the practice of the invention. Such genes encode enzymes that can convert a relatively non-toxic producing into a highly toxic agent. Cells genetically engineered to express such genes essentially commit metabolic suicide in the presence of appropriate prodrug.
In an embodiment of the invention, the herpes simplex thymidine kinase (HSV-TK) gene may be engineered into the recombinant retroviral vectors. Any cells subsequently infected with the recombinant retroviruses, and expressing the HSV-TK gene, would become sensitive to chemotherapeutic agents such as acyclovir and ganciclovir. In another embodiment of the invention the cytosine deaminase (CD) gene may be engineered into recombinant retroviral vectors. Cells expressing the CD gene metabolize the relatively non-toxic producing 5-flourocytosine to the highly toxic 5-fluorouracil (Mullen, C A et al., 1994, Cancer Res. 54:1503-6).
The method of the present invention further comprises the culturing of the producer cells in vitro on a support matrix prior to implantation into the mammalian brain. The preadhesion of cells to microcarriers prior to implantation in the brain is designed to enhance the long-term viability of the transplanted cells and provide long term functional benefit.
The invention is based, in part, on the demonstration that preadhesion of producer cells to microcarriers prior to transplantation into the mammalian brain enhances the viability of the transplanted cells. In a particular embodiment, described herein, producer cells were transplanted into the brains of rats. The transplanted producer cells produce infectious retrovirus particles that have been genetically engineered to express the alkaline phosphatase gene. Results demonstrate the successful long term expression of the alkaline phosphatase gene in the brain of the transplanted animal.